Quantification of cellular deoxynucleoside triphosphates (dNTPs).

Samples were extracted by adding 90 µl of an ice-cold 60% solution of methanol (aqueous) which contained 0.1 µg/ml of ¹³C10-¹⁵N5 ATP (Sigma-Aldrich) as an internal standard. Each sample was vortexed for 10 s and chilled at −20°C prior to being centrifuged for 10 min at 20,000 relative centrifugal force (RCF) at 4°C. The supernatant was removed to a fresh tube and dried overnight en vacuo. Immediately prior to liquid chromatography-mass spectrometry (LC-MS) analysis, the samples were reconstituted in 50 µl water containing 10 mM ammonium carbonate.

Mass spectral analysis was performed using an Agilent 6490 ultraperformance liquid chromatography (UPLC) triple-quadrupole (QQQ) MS system (Santa Clara, CA). A SeQuant ZIC hydrophilic interaction liquid chromatographic (HILIC) column (Millipore Sigma, Burlington, MA) was used for fractionation using a linear gradient of 10:90 acetonitrile (ACN)–10 mM ammonium acetate buffer to 20:80 ACN–10 mM ammonium acetate buffer over 20 min at a flow rate of 0.2 ml/min. Mass spectrometry was performed in the positive mode with an Agilent jet stream (AJS) electrospray ionization (ESI) source using tandem mass spectrometry (MS/MS) fragmentation. The deoxynucleotides were positively identified by both chromatographic retention time, and the ion ratios were identified by at least two selected reaction monitoring (SRM) transitions. Quantitative data analysis was conducted using Agilent MassHunter Quant software for dCTP using the internal standard to normalize signal between samples.